Abstract

XylT (beta1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

(A) Generation of two-chain A. thaliana XylT on prolonged storage. Purified recombinant A. thaliana XylT was stored in the absence (−) or presence (+) of proteinase inhibitors (E-64, leupeptin and PMSF) for 3 months at 4 °C. Samples were analysed by SDS/PAGE under reducing conditions and silver staining. The migration positions of selected molecular-mass standards are indicated. (B) The light chain of two-chain XylT is derived from the N-terminal part of the native enzyme. Purified recombinant A. thaliana XylT stored in the absence of proteinase inhibitors for 3 months was incubated overnight in the presence (+) or absence (−) of PNGase F. Samples were then analysed by SDS/PAGE under reducing conditions and silver staining or subjected to Western-blot analysis with anti-enterokinase recognition site antibodies. *, the migration position of PNGase F.

All three potential N-glycosylation sites of A. thaliana XylT are modified with N-linked oligosaccharides

The two-chain form of purified recombinant A. thaliana XylT was incubated overnight in the absence (A, C) or presence (B) of PNGase F before SDS/PAGE analysis. Bands corresponding to the XylT heavy chain were excised and subjected to trypsin treatment (A, B) or trypsin/endoproteinase Glu-C (C) treatment. Peptides thus generated were analysed by MALDI–TOF-MS. (A, B) The peptide N301FTKPVCFR309 is modified at Asn301 with N-linked oligosaccharides (A). The GP is partially modified at its cysteine residue with acrylamide instead of being carbamidomethylated, resulting in double peaks. The corresponding unglycosylated peptide was not detected in the sample. The N-glycan structures found were MM [Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc], MMX {Xylβ1-2[Manα1-6(Manα1-3)]Manβ1-4GlcNAcβ1-4GlcNAc} and MMF6 [Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc]. These GP peaks are absent from the deglycosylated sample (B). (C) The peptide A459SVIIGAHGAGLTHIVSATPN479TTIFE484 is modified at Asn479 with N-linked oligosaccharides. Two main N-glycan structures could be detected (MM and MMX). The corresponding unglycosylated peptide was not detected in the sample.

Sf21 insect cells infected with recombinant baculoviruses encoding Δ39 XylT were cultured in the absence (–) or presence (+) of tunicamycin. Cell extracts were then incubated overnight in the presence (+) or absence (–) of PNGase F before SDS/PAGE and immunoblot analysis with anti-enterokinase recognition site antibodies. The migration positions of selected prestained molecular-mass standards are indicated.

(A) Purified recombinant A. thaliana XylT was incubated with the indicated acceptor substrates and UDP-[14C]xylose. The samples were then analysed by TLC before orcinol/H2SO4 staining (lanes 1–5) and autoradiography (lanes 6 and 7). The migration direction is indicated by an arrow. (B) Purified recombinant human GnT II was incubated with the indicated acceptor substrates and UDP-[14C]GlcNAc. The samples were then analysed by TLC before orcinol/H2SO4 staining (lanes 1–5) and autoradiography (lanes 6 and 7).